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Hamster tracheal organ culture in serum-free media: A quantitative comparison of in vitro epithelial morphology with that of in vivo controls

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Summary

The epithelial morphology of the hamster trachea in serum-free organ culture was compared with that of age-matched in vivo control tissues by collecting and statistically analyzing several quantifiable parameters. By this technique it was possible to detect both subtle and dramatic epithelial alterations. Midtracheal tissues from 6-wk-old male Syrian golden hamsters were used as the explants. Explants were placed on Gelfoam sponges and cultured for 1, 2, and 3 wk in CMRL 1066 alone and in CMRL 1066 to which seven factors were added: insulin and transferrin (5 μg/ml); hydrocortisone (5×10−7 M); epidermal growth facotr (5 ng/ml); bovine pituitary extract (0.5%); and phosphoethanolamine and ethanolamine (5×10−5 M). The following data were collected and statistically analyzed for each tracheal ring: number of epithelial cells; proportion and number of each cell type; basement membrane length; linear density of epithelial cells; epithelial height; and mitotic index.

Compared to controls, ciliated cells decreased by 52% during washes in Leibovitz (L15) medium and tissue manipulation performed before culture and this loss persisted after cutlure for 1 wk. Explants culturedwithout the factors showed marked changes after 2 and 3 wk including epithelial thickening and folding, which was associated with increased linear density. Many cells in these specimens could not be categorized by type (22% were unidentifiable after 3 wk). Epithelial migration onto the outside of the explant was inhibited. In contrast, explants culturedwith the factors maintained a morphology similar to controls at 2 and 3 wk and epithelial migration onto the outside of the explant was supported. This study shows that explants in CMRL 1066 with the seven factors provide a useful biological model for the in vitro study of the mucociliary respiratory epithelium.

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References

  1. Asmundsson, T.; Kilburn, K. H.; McKenzie, W. N. Injury and metaplasia of airway cells due to SO2. Lab. Invest. 29:41–51; 1973.

    PubMed  CAS  Google Scholar 

  2. Barnes, D.; Sato, G. Serum-free culture: a unifying approach. Cell 22:649–655; 1980.

    Article  PubMed  CAS  Google Scholar 

  3. Barrett, L. A.; McDowell, E. M.; Frank, A. L., et al. Long-term organ culture of human bronchial epithelium. Cancer Res. 36:1003–1010; 1976.

    PubMed  CAS  Google Scholar 

  4. Becci, P. J.; McDowell, E. M.; Trump, B. F. The respiratory epithelium. II. Hamster trachea, bronchus, and bronchioles. JNCI 61:551–561; 1978.

    PubMed  CAS  Google Scholar 

  5. Bertolero, F.; Kaighn, M. F.; Gonda, M. A., et al. Mouse epidermal keratinocytes. Clonal proliferation and response to hormones and growth factors in serum-free medium. Exp. Cell Res. 155:64–80; 1984.

    Article  PubMed  CAS  Google Scholar 

  6. Bils, R. F.; Christie, B. R. The experimental pathology of oxidant and air pollutant inhalation. Int. Rev. Exp. Pathol. 21:195–293; 1980.

    PubMed  CAS  Google Scholar 

  7. Boatman, E. S.; Sato, S.; Frank, R. Acute effects of ozone on cat lungs. II. Structural. Am. Rev. Respir. Dis. 110:157–169; 1974.

    CAS  Google Scholar 

  8. Boren, H. G.; Pauley, J.; Wright, E. C., et al. Cell populations in the hamster tracheal epithelium in relation to vitamin A status. Int. J. Vitam. Nutr. Res. 44:382–390; 1974.

    PubMed  CAS  Google Scholar 

  9. Boren, H. G.; Paradise, L. J. Cytokinetics of lung In: Harris, C. C., ed. Pathogenesis and therapy of lung cancer. New York: Marcel Dekker; 1978:369–418.

    Google Scholar 

  10. Cherry, J. D. Mycoplasma pathogenicity studies in organ cultures. Ann. NY Acad. Sci. 225:290–303; 1973

    Article  Google Scholar 

  11. Chopra, D. P. Cell dynamics in explants derived from tracheas of hamsters fed normal and vitamin A-deficient diets. Cell Tissue Kinet. 16:155–165; 1983.

    PubMed  CAS  Google Scholar 

  12. Chopra, D. P. Retinoid reversal of squamous metaplasia in organ cultures of tracheas derived from hamsters fed on vitamin A-deficient diet. Eur. J. Cancer Clin. Onol. 19:847–857; 1983.

    Article  CAS  Google Scholar 

  13. Chopra, D. P.; Cooney, R. A. Histogenesis of benzo(a)pyreneinduce lesions in tracheal explants. Virchows Arch. [Cell Pathol.] 48:299–315; 1985.

    CAS  Google Scholar 

  14. Clark, J. N.; Marchok, A. C. The effect of vitamin A on cellular differentiation and mucous glycoprotein synthesis in long-term rat tracheal organ cultures. Differentiation 14:175–183; 1979.

    Article  PubMed  CAS  Google Scholar 

  15. Clark, J. N.; Klein-Szanto, A. J. P.; Pine, A. H., et al. Reestablishment of a mucociliary epithelium in tracheal organ cultures exposed to retinyl acetate: a biochemical and morphometric study. Eur. J. Cell Biol. 21:261–268; 1980.

    PubMed  CAS  Google Scholar 

  16. Clyde, W. A. Experimental models for study, of common respiratory viruses. Environ. Health Perspect. 35:107–112; 1980.

    Article  PubMed  Google Scholar 

  17. Collier, A. M. Injury of respiratory epithelium. Environ. Health Persp. 35:83–88; 1980.

    Article  CAS  Google Scholar 

  18. Cornillie, F. J.; Lauweryns, J. M.; Corbeel, L. Atypical bronchial cilia in children with recurrent repiratory tract infections. Pathol. Res. Pract. 178:595–604; 1984.

    PubMed  CAS  Google Scholar 

  19. Crocker, T. T.; Sanders, L. L. Influence of vitamin A and 3, 7-dimethyl-2,6-octadienal (citral) on the effect of benzo(a)pyrene on hamster trachea in organ culture. Cancer Res. 30:1312–1318; 1970.

    PubMed  CAS  Google Scholar 

  20. Dahlgren, S. E.; Dalen, H. Ultrastructural observations on chemically induced inflammation in guinea pig trachea. Virchows Arch. [Cell Pathol.] 11:211–223; 1972.

    CAS  Google Scholar 

  21. Daniel, P. F.; Wolf, G. Incorporation of labeled glucosamine into glycoproteins by organ cultures of hamster trachea: adverse effects of HEPES buffer. In Vitro 11:347–353; 1975.

    Article  PubMed  CAS  Google Scholar 

  22. Daniel, W. W. Biostatistics: a foundation for analysis in the health sciences. New York: John Wiley and Sons; 1983:224–225.

    Google Scholar 

  23. De Brabander, M.; De May, J.; Geuens, G., et al. Microtubules in cell organization and motility. In: Dumont, J. E.; Hamprecht, B. Nunez, J., eds. Hormones and cell regulation, vol. 9. Amsterdam: Elsevier Science Publishing; 1985:85–104.

    Google Scholar 

  24. Dirksen, E. R.; Crocker, T. T. Ultrastructural alterations produced by poloycyclic aromatic hydrocarbons on rat tracheal epithelium in organ culture. Cancer Res. 28:906–923; 1968.

    PubMed  CAS  Google Scholar 

  25. Feron, V. J.; Immel, H. R.; Spit, B. J. Restoration of the hamster tracheal wall following injury by electrocoagulation. Exp. Mol. Pathol. 41:236–248; 1984.

    Article  PubMed  CAS  Google Scholar 

  26. Fujikawa, L. W.; Foster, C. S.; Gipson, I.K., et al. Basement membrane components in healing rabbit corneal epithelial wounds: immunofluorescence and ultrastructural studies. J. Cell Biol. 98:128–138; 1984

    Article  PubMed  CAS  Google Scholar 

  27. Ham, R. G. Formulation of basal nutrient media. In: Barnes, D. W.; Sirbasku, D. A.; Sato, G. H., eds. Methods for preparation of media, supplements, and substrata for serumfree animal cell culture. New York: Alan R. Liss Inc.; 1984:3–21.

    Google Scholar 

  28. Harris, C. C.; Sporn, M. B.; Kaufman, D. G., et al. Acute ultrastructural effects of benzo(a)pyrene and ferric oxide on the hamster tracheobronchial epithelium. Cancer Res. 31:1977–1979; 1971.

    PubMed  CAS  Google Scholar 

  29. Hayashi, I.; Sato, G. H. The replacement of serm by hormones permits the growth of cells in a defined medium. Nature 259:132–134; 1976.

    Article  PubMed  CAS  Google Scholar 

  30. Heckman, C. A.; Marchok, A. C.; Nettesheim, P. Respiratory tract epithelium in primary culture: concurrent growth and differentiation during establishment. J. Cell Sci. 32:269–291; 1978.

    PubMed  CAS  Google Scholar 

  31. Jockusch, B. M.; Füchtbauer, A.; Wiegland, C., et al. Dynamics of microfilament organization and membrane anchorage. In: Dumont, J. E.; Hamprecht, B.; Nunez, J., eds. Hormones and cell regulation, vol. 9. Amsterdam: Elsevier Science Publishing; 1985:135–141.

    Google Scholar 

  32. Joens, R. T.; Bertolero, F.; Kaighn, M. E., et al. Optimal conditions for primary epithelial cell outgrowth culture of adult hamster trachea. In Vitro 20:270; 1984.

    Google Scholar 

  33. Keenan, K. P.; Combs, J. W.; McDowell, E. M. Regenration of hamster tracheal epithelium after mechanical injury. I. Focal lesions: quantitative morphologic study of cell proliferation. Virchows Arch. [Cell Pathol.] 41:193–214; 1982.

    CAS  Google Scholar 

  34. Keenan, K. P.; Combs, J. W.; McDowell, E. M. Regeneration of hamster tracheal epithelium after mechanical injury. III. Large and small lesions: comparative stathmokinetic and single pulse and continuous thymidine labeling autoradiographic studies. Virchows Arch. [Cell Pathol.] 41:231–252; 1982.

    CAS  Google Scholar 

  35. Keenan, K. P.; Wilson, T. S.; McDowell, E. M., Regeneration of hamster tracheal epithelium after mechanical injury. IV. Histochemical immunocytochemical, and ultrastructural studies. Virchows Arch. [Cell Pathol.] 43:213–240; 1983.

    CAS  Google Scholar 

  36. Kennedy, A. R; Desrosiers, A.; Terzaghi, M., et al. Morphometric and histological analysis of the lungs of Syrian golden hamster. J. Anat. 125:527–533; 1978.

    PubMed  CAS  Google Scholar 

  37. Kidwell, W. R.; Bano, M.; Salomon, D. S. Growth of normal mammary epithelium on collagen in serum-free medium. In: Barnes, D. W.; Sirbasku, D. A.; Sato, G. H., eds., Methods for serum-free culture of cells of the endocrine system. New York: Alan R. Liss, Inc.; 1984:105–125.

    Google Scholar 

  38. Lane, B. P.; Miller, S. L. Preparation of large numbers of uniform tracheal organ cultures for long-term studies I. Effects of serum on establishment in culture. In Vitro 12:147–154; 1976.

    PubMed  CAS  Google Scholar 

  39. Lane, B. P.; Miller, S. L. Carcinogen-induced changes in tracheal epithelium cultured in serum-free, chemically defined medium. JNCI 56:991–996; 1976.

    PubMed  CAS  Google Scholar 

  40. Lechner, J. F.; Haugen, A.; McClendon, I. A., et al. Clonal growth of normal adult human bronchial epithelial cells in a serum-free medium. In Vitro 18:633–642; 1982.

    PubMed  CAS  Google Scholar 

  41. Lechner, J. F.; McClendon, I. A.; LaVeck, M. A., et al. Differential control by platelet factors of squamous differentiation in normal and malignant human bronchial epithelial cells. Cancer Res. 43:5915–5921; 1983.

    PubMed  CAS  Google Scholar 

  42. Lee, T.-C.; Wu, R.; Brody, A. R., et al. Growth and differentiation of hamster tracheal epithelial cells in culture. Exp. Lung Res. 6:27–45; 1984.

    PubMed  CAS  Google Scholar 

  43. Lloyd, C.; Kennedy, J. R.; Mendicino, J. Regulation of the synthesis of mucin glycoproteins in swine trachea explants. In Vitro 20:416–433; 1984.

    Article  PubMed  CAS  Google Scholar 

  44. Marchok, A. C.; Cone, M. V.; Nettesheim, P. Induction of squamous metaplasia (vitamin A deficiency) and hypersecretory activity in tracheal organ cultures. Lab. Invest. 33:451–460; 1975.

    PubMed  CAS  Google Scholar 

  45. McCarthy, J. B.; Basara, M. I.; Palm., S. L., et al. The role of cell adhesion proteins—laminin and fibronectin—in the movement of malignant and metastatic cells. Cancer Metastasis Rev. 4:125–152; 1985.

    Article  PubMed  CAS  Google Scholar 

  46. McDowell, E. M.; Barrett, L. A.; Harris, C. C., et al. Abnormal cilia in human bronchial epithelium. Arch. Pathol. Lab. Med. 100:429–436; 1976.

    PubMed  CAS  Google Scholar 

  47. McDowell, E. M.; Trump, B. F. Histologic fixatives suitable for diagnostic light and electron microscopy. Arch. Pathol. Lab. Med. 100:405–414; 1976.

    PubMed  CAS  Google Scholar 

  48. McDowell, E. M.; Barrett, L. A.; Glavin, F., et al. The respiratory epithelium. I. Human bronchus. JNCI 61:539–549; 1978.

    PubMed  CAS  Google Scholar 

  49. McDowell, E. M.; Becci, P. J.; Scürch, W., et al. The respiratory epithelium. VII. Epidermoid metaplasia of hamster tracheal epithelium during regeneration following mechanical injury. JNCI 62:995–1008; 1979.

    PubMed  CAS  Google Scholar 

  50. McDowell, E. M.; Combs, J. W.; Newkirk, C. A quantitative light and electron microscopic study of hamster tracheal epithelium with special attention to so-called intermediate cells. Exp. Lung Res. 4:205–226; 1983.

    PubMed  CAS  Google Scholar 

  51. McDowell, E. M.; Trump, B. F. Histogenesis of preneoplastic and neoplastic lesions in tracheobronchial epithelium. Surv. Synth. Path. Res. 2:235–279; 1983.

    Google Scholar 

  52. McDowell, E. M.; Keenan, K. P.; Huang, M. Effects of vitamin A-deprivation on hamster tracheal epithelium. Virchows Arch. [Cell Pathol.] 45:197–219; 1984.

    CAS  Google Scholar 

  53. McDowell, E. M.; Keenan, K. P.; Huang, M. Restoration of mucociliary tracheal epithelium following deprivation of vitamin A: a quantitative morphologic study. Virchows Arch. [Cell Pathol.] 45:221–240; 1984.

    Article  CAS  Google Scholar 

  54. McKeehan, W. L.; Hamilton, W. G.; Ham, R. G. Selenium is an essential trace nutrient for growth of WI-38 diploid human fibroblasts. Proc. Natl. Acad. Sci. USA 73:2023–2027; 1976.

    Article  PubMed  CAS  Google Scholar 

  55. Mossman, B. T.; Craighead, J. E. Long-term maintenance of differentiated respirtory epithelium in organ culture. I. Medium composition. Proc. Soc. Exp. Biol. Med. 149:227–233; 1975.

    PubMed  CAS  Google Scholar 

  56. Mossman, B. T.; Kessler, J. B.; Ley, B. W., et al. Interaction of crocidolite asbestos with hamster respiratory mucosa in organ culture. Lab. Invest. 36:131–139; 1977.

    PubMed  CAS  Google Scholar 

  57. Mossman, B. T.; Heintz, N.; Macpherson, B. V., et al. Squamous metaplasia of the tracheal epithelium in organ culture. II. Nutritional influences. Proc. Soc. Exp. Biol. Med. 157:500–505; 1978.

    PubMed  CAS  Google Scholar 

  58. Muse, K. E.; Collier, A. M.; Baseman, J. B. Scanning electron microscopic study of hamster organ cultures infected withBordetella pertussis. J. Infect. Dis. 136;768–777;1977.

    PubMed  CAS  Google Scholar 

  59. Newton, D. L.; Henderson, W. R.; Sporn, M. B. Structureactivity relationships of retinoids in hamster tracheal organ culture. Cancer Res. 40:3413–3425; 1980.

    PubMed  CAS  Google Scholar 

  60. Otani, E. M. The basal cell progenitor of the tracheobronchial epithelium: fact or folklore? In: McDowell, E. M., ed. Lung carcinomas. Edinburgh: Churchill Livingstone; In press.

  61. Palekar, L.; Kuschner, M.; Laskin, S. The effect of 3-methylcholanthrene on rat trachea in organ culture. Cancer Res. 28:2098–2104; 1968.

    PubMed  CAS  Google Scholar 

  62. Resau, J. R.; Jones, R. T.; Hudson, E. A. Organ explant culture of adult Syrian golden hamster pancreas. In Vitro 19:315–325; 1983.

    Article  PubMed  CAS  Google Scholar 

  63. Ruddell, C. L. Epoxy media for 1–2 micron sectioning. Stain Technol. 42:253–255; 1967.

    PubMed  CAS  Google Scholar 

  64. Schiff, L. J.; Byrne, M. M. Fly ash-induced changes in hamster tracheal epitheliumin vivo andin vitro. J. Toxicol. Environ. Health 8:431–448; 1981.

    Article  PubMed  CAS  Google Scholar 

  65. Schiff, L. J.; Moore, D. Effects of cyclic adenosine 3′:5′-monophosphate-elevating agents and retinoic acid on differentiation in retinoid-deficient tracheal cultures. In Vitro 21:688–692; 1985.

    CAS  Google Scholar 

  66. Snyder, C. E.; Nadzieko, C. E.; Herp, A. Human bronchial explants in long-term culture: establishing a baseline for secretion. In Vitro 20:95–102; 1984.

    Article  PubMed  CAS  Google Scholar 

  67. Soong, H. K.; Cintron, C. Different corneal epithelial healing mechanisms in rat and rabbit: role of actin and calmodulin. Invest Ophthalmol. Vis. Sci. 26:838–848; 1985.

    PubMed  CAS  Google Scholar 

  68. Sorokin, S. P.; Hoyt, R. F. PAS-lead hematoxylin as a stain for small-granule endocrine cell populations in the lungs, other pharyngeal derivatives and the gut. Anat. Rec. 192:245–249; 1978.

    Article  PubMed  CAS  Google Scholar 

  69. Sporn, M. B.; Clamon, G. H.; Dunlop, N. M., et al. Activity of vitamin A analogues in cell cultures of mouse epidermis and organ cultures of hamster trachea. Nature 253:47–49; 1975.

    Article  PubMed  CAS  Google Scholar 

  70. Strum, J. M.; Latham, P. S.; Schmidt, M. L., et al. Vitamin A deprivation in hamsters. Correlations between tracheal epithelial morphology and serum/tissue levels of vitamin A. Virchows Arch. [Cell Pathol.] 50:43–57; 1985.

    CAS  Google Scholar 

  71. Torikata, C.; Tekeuchi, H.; Yamaguchi, H., et al. Abnormal cilia in the bronchial mucosa. Virchows Arch. [A] 371:121–129; 1976.

    Article  CAS  Google Scholar 

  72. Wu, R.; Smith, D. Continuous multiplication of rabbit tracheal epithelial cells in a defined hormone-supplemented medium. In Vitro 18:800–812; 1982.

    PubMed  CAS  Google Scholar 

  73. Wu, R.; Groelke, J. W.; Chang, L. Y., et al. Effects of hormones on the multiplication and differentiation of tracheal epithelial cells in culture. In: Sirbasku, D.; Sato, G. H.; Pardee, A., eds. Growth of cells in hormonally defined media. vol. 9. New York: Cold Spring Harbor; 1982:641–656.

    Google Scholar 

  74. Wu, R.; Yankaskas, J.; Cheng, E., et al. Growth and differentiation of human nasal epithelial cells in culture. Am. Rev. Respir. Dis. 132:311–320; 1985.

    PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Pathology, University of Maryland School of Medicine, 10S, Pine Street, 21201, Baltimore, Maryland

    Robert E. Sigler, Raymond T. Jones & Elizabeth M. McDowell

  2. Department of Epidermiology and Preventive Medicine, University of Maryland School of Medicine 10 S, Pine Street, 21201, Maryland, Baltimore

    John R. Hebel

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This work was supported by grant HL24722 from the National Institutes of Health, Bethesda, Maryland.

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Sigler, R.E., Jones, R.T., Hebel, J.R. et al. Hamster tracheal organ culture in serum-free media: A quantitative comparison of in vitro epithelial morphology with that of in vivo controls. In Vitro Cell Dev Biol 23, 100–110 (1987). https://doi.org/10.1007/BF02623589

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